Biomaterials have been applied extensively to the urogenital area of children and adults. For example, in 1992, approximately, 58 billion diapers, 74 billion tampons, and 16 million urinary catheters were used in North America alone. Of course, other biomaterials have also been used in other areas of the body as well as in the urogenital tract, such as stents, fibrous materials, diaphragms and the like. Unfortunately, the adhesion of bacteria to the surfaces of these biomaterials is one mechanism whereby pathogenic bacteria can form a nidus for infection. This is particularly important as there are many microorganisms in the urogenital tract to which these materials are exposed. For example, it has been shown that pathogenic bacteria bind to catheters, stents, tampons and fibrous material, leading to various urogenital infections. Therefore, there is a need to find some product that will reduce the risk of contracting infections and particularly urogenital infections.
It has been found that the urogenital tract of the healthy pre-menopausal female is dominated by lactobacilli, while the urogenital microflora of women suffering recurrences of urinary tract infection (UTI) is replaced almost entirely by uropathogens. In fact, facultative lactobacilli make up 50-90% of the aerobic vaginal microflora in premenopausal women, and are also abundant in the aerobic urethral flora of healthy women in the reproductive age, accounting for 38% of the aerobic flora. In vitro, animal and human studies have provided evidence that indigenous lactobacilli may protect the host against urinary tract infection.
Lactobacilli are able to interfere with uropathogenic bacteria through several mechanisms. Lactobacillus whole cells and cell wall fragments have been found to competitively exclude a range of uropathogens from adhering to uroepithelial cells (Chan, et al. (1985) Infect. Immun. 49:84-89; Reid, et al. (1987) J. Urol. 138:330-335). competitive exclusion of uropathogens from attaching to polymer and catheter surfaces by lactobacilli has also been demonstrated (Hawthorn, et al. (1990) J. Biomed. Mater. Res. 24:39-46; Reid and Tieszer (1993) Cells and Materials 3:171-176). Lactobacilli have also been shown to coaggregate with uropathogenic bacteria which, in combination with inhibitor production, may lead to elimination of the uropathogens from surfaces (Reid, et al. (1988) Can. J. Microbiol. 34:344-351). Lactobacilli are also known to produce a variety of metabolic by-products with antimicrobial activity, such as hydrogen peroxide, lactic acid, bacteriocins and bacteriocin-like substances. However, prior to the present invention, no one identified the biosurfactant substances produced by the lactobacilli that were responsible for inhibiting the adhesion of pathogenic and particularly uropathogenic bacteria. As described hereinbelow, the present inventors have identified that substance, isolated it, and discovered that this substance is important for the inhibitory effects described hereinabove.
Another major problem associated with biomaterial devices, especially catheters, is solved by the present invention. In relation to infection, the insertion of urethral catheters is perhaps best recognized for an association with not only urinary tract infections (UTI) but also bacteremia and sepsis. The inability to eradicate the infecting organisms appears, in many cases, to be due to failure of antimicrobial therapy to penetrate biofilms. Only by removing the device does the patient respond, temporarily in some cases, to drug treatment. The biofilm problem extends to many other areas including devices used in urological, nephrological, anesthetic, respiratory, cardiovascular, general surgical and orthopedic practice, for example.
A microbial biofilm is defined as an accumulation of microorganisms and their extracellular products to form a structured community usually on a surface. More recently, the term has been broadened to include biofilms at some distances away from a surface (e.g. in disease states such as prostatitis), and which exist in multiple as well as single layers of cells.
The formation of an infectious biofilm on biomaterials consists of several sequential steps, and includes the deposition of the infectious microorganisms, adhesion of the organisms, anchoring by exopolymer production and growth of the organisms.
Immediately after insertion of a device into the body, the material surface is contacted with body fluids, such as saliva, tear fluid, blood or urine, for example. Macromolecular components from these body fluids adsorb quickly onto the material surfaces to form a conditioning film, prior to the arrival of the first organisms. The deposition of such conditioning films has been demonstrated on surfaces such as urinary catheters and ureteral stents. The compositions of these conditioning films have not been specifically defined, but nitrogen, carbon, oxygen, calcium, sodium and phosphorous have been identified as composing elements by x-ray photoelectron-spectroscopy and energy dispersive x-ray analysis.
The importance of the conditioning film and the initially adhering microorganisms have long been underestimated. This is, in part, due to the fact that the subsequent growth of the organisms leads to the dense biofilms, eventually manifesting in a clinical problem. However, the important first link in the chain of events leading to the formation of mature biofilms involves the initially adhering organisms. Accordingly, this bond represents the link with the growing biofilm. If this linkage breaks, the formation of the biofilms is either prevented or the formed biofilm detaches, thereby aiding the eradication of infection.
Again, the present inventors have discovered a substance that inhibits biofilm formation. In fact, this substance is the same substance that inhibited the adhesion of the uropathogenic bacteria. This substance, which the present inventors have found is a biosurfactant of lactobacilli.
Biosurfactants are compounds released by various microorganisms including lactobacilli, with a distinct tendency to accumulate at interfaces, most notably the liquid-air interface. Biosurfactant production can be measured conventionally by axisymmetric drop shape analysis by profile (ADSA-P). Classes of biosurfactants can be distinguished, according to their chemical structure. The most extensively investigated biosurfactants are glycolipids, e.g. the rhamnolipids from Pseudomonas aeruginosa. Other types of biosurfactants are lipopeptides and protein-like substances, phospholipids, substituted fatty acids, and lipopolysaccharides. The biosurfactants produced by these bacteria have different functions. For example, dairy Streptococcus thermophilus can produce biosurfactants which cause their own desorption, and oral Streptococcus mitas strains produce biosurfactants with anti-adhesive properties against Streptococcus mutans.
Various physiological functions of biosurfactants have also been described. Biosurfactants can, inter alia, enable microorganisms to grow on water-immiscible compounds by lowering the surface tension at the phase boundary; biosurfactants can cause emulsification, and can stimulate adhesion of microbial cells to organic substrates.
Biosurfactants have advantages over synthetic surfactants and it is those advantages that make biosurfactants prime candidates for industrial and biomedical applications. Biosurfactants are biodegradable and those from lactobacilli are non-toxic to humans.
Heretofore, no one knew that lactobacilli produced biosurfactants. However, the present inventors have not only discovered that lactobacilli produce biosurfactants, but also have isolated same and have discovered that these isolated biosurfactants can be used to prevent biofilm formation and urogenital infections.